The invention relates to an optical evaluation method by means of laser pulses as well as to a corresponding apparatus.
A large number of optical measuring methods is known in which a sample interacts with laser light from different laser sources (excitation light), and as a result of this interaction an optical signal, for example, light of a different color or wavelength, polarization, intensity or the like, is generated and can be detected. Due to the position-dependent detection of the detection light, such methods can be used inter alia for imaging, for example in microscopy. Examples thereof are mainly methods which make use of frequency doubling, frequency multiplication, sum frequency mixing, difference frequency mixing, four-wave-mixing, CARS (Coherent anti-Stokes Raman Spectroscopy), FM-CARS, OCT, stimulated fluorescence, stimulated Raman scattering, i.e. stimulated Raman gain or stimulated Raman loss etc.
From Chem. Phys. Lett. 2007, 442, 483-487, an optical evaluation method is known, in which the effect of stimulated fluorescence is made use of. Here, fluorescent dyes are first placed in an excited state by a first laser pulse. When a second laser pulse of a suitable wavelength is irradiated within a very short time lag, i.e. within some nanoseconds, this second excitation laser pulse is amplified by the excited dyes, and a detection light is generated which, however, due to the same wavelength can only hardly be distinguished from the excitation light. On a suitable detector, an optical signal is detected which, given an incidence of the two laser pulses at the sample within a very short time lag, differs from the signal which would be generated when both laser pulses would not be incident on the sample within a very short time lag. In this specific case, the first laser pulse is already suppressed upstream of the detector by means of suitable spectral filters so that exclusively the second laser pulse as well as the additionally generated optical signal arrive at the detector. In order to only obtain the desired additional optical signal, one would have to make a measurement with the first excitation laser pulse and a measurement without the first excitation laser pulse, and to form the respective difference. However, the two measurements differ from one another so little (in the order of less than 10−8) that due to the limited dynamic range of the detectors or due to detection noise etc. the desired optical signal can no longer be perfectly detected. Therefore, in such cases as well as in the cited publication usually the known lock-in technology is used with the aid of which the desired signal can still be separated from the background.
A disadvantage of this lock-in technology is, however, the limited speed which represents a clear disadvantage in particular for imaging methods such as laser scanning technologies. In particular, when used in laser scanning microscopy, where frame rates of up to 25 frames/second given image sizes of 512×512 pixels are common, wherein for individual pixels then only times of clearly less than 1 microsecond are available, the use of this technology would result in a clear slowing down of the image taking, which—at least for the examination of processes in living cells—is inacceptable.